Aβ42 is believed to play a causative role in Alzheimer’s disease (AD) pathogenesis. γ-Secretase modulators (GSMs) are actively being pursued as potential AD therapeutics because they selectively alter the cleavage site of the amyloid precursor protein (APP) to reduce the formation of Aβ42. However, the binding partner of acid based GSMs was unresolved until now. We have developed clickable photoaffinity probes based on piperidine acetic acid GSM-1 and identified PS1 as the target within the γ-secretase complex. Furthermore, we provide evidence that allosteric interaction of GSMs with PS1 results in a conformational change in the active site of the γ-secretase complex leading to the observed modulation of γ-secretase activity.
␥-Secretase cleaves multiple substrates within the transmembrane domain that include the amyloid precursor protein as well as the Notch family of receptors. These substrates are associated with Alzheimer disease and cancer. Despite extensive investigation of this protease, little is known regarding the regulation of ␥-secretase specificity. To discover selective inhibitors for drug development and for probing the mechanisms of ␥-secretase specificity, we screened chemical libraries and consequently developed a di-coumarin family of inhibitors that preferentially inhibit ␥-secretase-mediated production of A42 over other cleavage activities. These coumarin dimerbased compounds interact with ␥-secretase by binding to an allosteric site. By developing a multiple photo-affinity probe approach, we demonstrate that this allosteric binding causes a conformational change within the active site of ␥-secretase at the S2 and S1 sub-sites that leads to selective inhibition of A42. In conclusion, by using these di-coumarin compounds, we reveal a mechanism by which ␥-secretase specificity is regulated and provide insights into the molecular basis by which familial presenilin mutations may affect the active site and specificity of ␥-secretase. Furthermore, this class of selective inhibitors provides the basis for development of Alzheimer disease therapeutic agents. affinity labeling ͉ Alzheimer disease ͉ allosteric regulation ͉ di-coumarin ␥ -Secretase is a multi-protein membrane-bound complex that is currently at the front line of basic and translational research. It is composed of at least 4 proteins that include presenilin, nicastrin, Aph-1, and Pen-2 (1). Presenilin is believed to contain the active site of ␥-secretase (2-4). It represents a novel class of protease that catalyzes peptide bond hydrolysis within the transmembrane hydrophobic environment and plays an essential role in a newly emerged signaling pathway known as regulated intramembrane proteolysis (5). ␥-Secretase cleaves a variety of type I membrane proteins that include the amyloid precursor protein (APP) and the Notch family of proteins despite limited primary sequence homology across targeted substrates (6). Elucidation of the mechanisms that control the specificity of ␥-secretase for these substrates has been hindered by technical difficulties associated with intramembrane enzymology. Determining the factors that contribute to ␥-secretase specificity is critical to understanding the biology of this unique protease and targeting it for therapeutic purposes.␥-Secretase has become an appealing drug target for Alzheimer disease (AD) and cancer because of its central role in the processing of APP and Notch (6). ␥-Secretase cleaves APP to generate neurotoxic A peptides, ranging from 37 to 46 aa in length (7). Among them, A40 and A42 have been extensively investigated for their association with AD (7). Additionally, diseasecausing familial AD mutations within APP, presenilin-1 (PS-1), and presenilin-2 (PS-2) proteins result in an increase in the ratio of A42...
Background:The effect of PS1 mutations on the active site of ␥-secretase is unknown. Results: PS1 mutations reduce photoprobe interaction with the S2 subsite of ␥-secretase and Notch1 cleavage. Conclusion: Certain PS mutations specifically alter the S2 subsite of ␥-secretase active site that leads to changes in APP and Notch1 cleavage. Significance: It provides structural insights into the mechanism of PS1 mutations in regards to ␥-secretase regulation.
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